The isolation and analysis of nucleic acids from various sources is a commonly performed procedure in genetic and recombinant DNA research. As the primary genetic elements, nucleic acids will exist in various forms depending on the biological source: Mammalian sources (such as blood) contain large, double-stranded, filamentous DNA (20-500 million bases); viruses (HIV or Epstein-Barr) can contain single- or double-stranded DNA or RNA, filamentous or closed-circular in structure (10-200 kilobases); bacteria (particularly variants of E. coli K-12) contain a single chromosome (4 million bases) and extra chromosomal elements, either plasmids or cosmids (2-50 kilobases), in E. coli these elements are all double-stranded, closed-circular DNA molecules. The procedures and chemistries commonly employed T. Maniatis et al.: Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory (1989).
The chemistries typically involve breaking open the cells or viral particles, extracting cellular and protein contaminants, purifying and concentrating the nucleic acids and then resuspending it in small volume prior to use. Purification of plasmids or cosmids requires the additional step of separation from the bacterial chromosome prior to their use. This technique, originally described by H. C. Birnboim and J. Doly "A Rapid Alkaline Extraction Procedure for Screening Recombinant Plasmid DNA", Nucleic Acids Research 7:1513-1523 (1979), accomplishes this separation based on the plasmid or cosmid's capacity to resist denaturation (i.e. separation of the complementary DNA strands into their single-stranded components). Since the size of the DNA molecule influences its resistance to denaturation, and subsequent co-purification with plasmids and cosmids, it is important that the bacterial chromosome not be broken into smaller fragments during the course of the procedure. For this reason, manual protocols emphasize gentle mixing. In general, any DNA isolation procedure that will reduce the amount of shearing of chromosomal DNA is desirable.
A major contributor to the breakup of large bacterial chromosomes are the shear forces generated during the preparational procedure. When the procedures are performed manually, the undesirable high shear forces are avoided by accomplishing mixing by slowly inverting the test tubes. However, in automated separation procedures employing automatic equipment to perform some or all of the separation steps, such slow, gentle mixing by capping and inverting the test tubes is difficult and costly to accomplish.
In automated DNA separation techniques, the tube contents are typically mixed by repeated pipetting of the solution, blowing bubbles into the solution, or shaking the tubes to simulate vortexing. Each of these mixing procedures generates significant shear forces as compared to the slow-inversion manual technique. Accordingly, the automated DNA separation procedures have been characterized by low yields of plasmid or cosmid DNA of an inferior quality, at least partially due to contamination by chromosomal fragments. In addition, the automated separation procedures typically take more time than the manual methods because the mixing steps are very slow and inefficient.